Methods to extract NAD+-malate dehydrogenase efficiently from white spruce needles

The spectrophotometrically-determined activity of NAD⁺-malate dehydrogenase (MDH, EC 1.1.1.37) from white spruce Picea glauca (Moench) Voss] needles was assayed with NADH and oxaloacetate. Activity was very low when extracted with only acetate buffer (pH 5.4), phosphate buffer (pH 6.8), or Tris-HCl buffer (pH 8.0). However, activity increased from 1 to over 200 μmol (g dry weight)^-1 min^-1 with the addition of polymers such as polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) and the detergents, Tween 80, Tergitol 15-S-9 and Triton X-100. Best activity was observed when extracted in a buffer at pH 6.8 and with 1% (v/v) for the three detergents and PEG, and 6% (w/v) for PVP.
MDH activity decreased with age of the needles on the tree. Six-year-old needles contained only about one-fifth of the activity of current year, fully-expanded needles. The main decrease in enzyme activity was observed in one-year-old needles. Protein content obtained from needles extracted with just phosphate buffer (pH 6.8) was very low, but increased greatly when the above chemicals were added to the buffer. In contrast with needles, extracts of vegetative buds contained much higher levels of MDH and protein when extracted with only phosphate buffer (pH 6.8). Although MDH activity in needle extracts declined with storage of the extracts at 4°C in the dark for 6 days, the decrease was least for buffers containing a combination of different protective agents.